An assessment of fungicide resistance in Venturia inaequalis populations: Mycelium growth analysis and molecular techniques

Kimberly Sue Chapman, Purdue University

Abstract

Apples are a high value commodity, and thus proper management of pests is required to produce high quality, marketable fresh fruit. The most economically important disease of apples is apple scab. Venturia inaequalis, the causal agent of apple scab, infects both commercial apples and ornamental crabapples and is primarily controlled by fungicides. Long-term and extensive fungicide use has led to decreased sensitivity of V. inaequalis to multiple fungicide modes of action. Detection and quantification of fungicide resistance is vital in allowing growers to make appropriate chemical management decisions. The development of fungicide resistance results in fewer management options for apple scab and the potential for increased economic and crop losses. To assess fungal sensitivity to fungicides, isolates of V. inaequalis were collected from Indiana and Michigan orchards. Single-spore derived isolates were evaluated using mycelium growth assays with previously determined fungicide baseline concentrations and corresponding thresholds for growth for dodine, kresoxim-methyl, myclobutanil and thiophanate-methyl. Of the 195 isolates tested, 5.2%, 0.7%, 57.0% and 92.6% of isolates were found to be resistant to dodine, kresoxim-methyl, myclobutanil and thiophanate-methyl, respectively. This is the first report of kresoxim-methyl resistance in the pathogen, field populations in both of these states. Isolates that tested resistant or shifted to a single fungicide were often found to have multiple fungicide resistance. Of all isolates tested from both states, 38% were identified as resistant or shifted to two fungicides and 12% were resistant or shifted to all four fungicides. Isolates resistant to multiple fungicides were found to have no fitness penalty based on statistical analysis of mycelium growth and conidial production. Fungicide resistance leaves fewer effective fungicide modes of action for apple scab management; however, one option may be difenoconazole, which was found to inhibit the growth of myclobutanil-resistant or shifted isolates. While mycelium growth assays can be used to determine the presence of fungicide resistant phenotypes in a population, results take months to generate and to further compound this problem, pure cultures of the pathogen cannot be obtained after the beginning of June. Therefore, molecular methods were developed for rapid detection of resistance, without first obtaining fungal cultures. Four classes of benzimidazole fungicide sensitivity were found in the Indiana V. inaequalis population: sensitive (S) isolates unable to grow on 0.5 &mgr;g active ingredient (a.i.)/ml; low resistant (LR) isolates that grew at 0.5 &mgr;g a.i./ml, but not at 5 &mgr;g a.i./ml; moderately resistant (MR) isolates that grew at 5 &mgr;g a.i./ml, but not at 50 &mgr;g a.i./ml; and very highly resistant (VHR) isolates that grew rapidly at 50 &mgr;g a.i./ml. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the β-tubulin gene with two restriction enzymes, BstUI and Cac8I, enabled us to rapidly identify benzimidazole resistance among all tested isolates. Sixty-nine percent of the resistant isolates tested possessed the BstUI RFLP at codon 198 that corresponds to VHR, and the remaining LR and MR isolates possessed the Cac8I RFLP corresponding to a newly identified resistance allele at codon L240F. Combined, PCR-RFLP correctly identified the resistance status of all isolates tested to date. The prevalence of benzimidazole-resistant isolates from commercial apple orchards and their absence in the landscape on ornamental crabapple suggests that two distinct populations of V. inaequalis coexist in Indiana.

Degree

M.S.

Advisors

Beckerman, Purdue University.

Subject Area

Plant Pathology

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